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1.  Resistance of Bacillus subtilis var. niger Spores Occluded in Water-insoluble Crystals to Three Sterilization Agents 
Applied Microbiology  1967;15(4):726-730.
The resistance to destruction of spores of Bacillus subtilis var. niger occluded in crystals of calcium carbonate and exposed to ethylene oxide and moist and dry heat was determined and compared with the destruction of unoccluded spores. Occluded spores could not be inactivated with ethylene oxide. Resistance to inactivation was approximately 900 and 9 times higher for occluded than for unoccluded spores subjected to moist and dry heat, respectively, at 121 C. The protective effect may be due either to the unavailability of oxygen for destruction by oxidation or to inhibition of the loss of essential cell constituents by vaporization. Evidence also implicates the crystal structure as a thermal conductivity barrier. Occluded spores retained viability over a 3-year period compared with unoccluded spores which decreased over 90% during this period. Occluded spores in insoluble materials are seldom encountered in the technology of sterilization, but could be the most critical factor in the sterilization of interplanetary vehicles. Entrapped spores in insoluble materials are usually difficult to detect, and are very stable as well as extremely resistant to destruction by heat and ethylene oxide.
PMCID: PMC547045  PMID: 4963439
2.  Mechanism of the Heat Sensitization of Bacillus subtilis Spores by Ethidium Bromide 
Pretreatment with ethidium bromide (5 μg/ml) followed by a water wash had no effect on unheated Bacillus subtilis spores, but the viability of these spores after heating was much lower than that of similarly heated spores exposed to water alone. The fate of water- or ethidium bromide-treated spores, unheated or heated, was followed by allowing them to germinate and outgrow in a minimal or a complex liquid medium. Spores exposed to ethidium bromide and then heated (85°C, 10 min) exhibited a developmental block during germination and outgrowth. Many of them were blocked at the stage when the bacterium emerged from the germinated spore. When 0.35 μg of ethidium bromide per ml was added to heated spores in the germination-growth medium, the outgrowth of heated spores was inhibited to the same extent as were pretreated spores. Ethidium bromide acted in the first hour of germination of heated spores since addition after this time was ineffective in inhibiting recovery events. Repair of heat-damaged spore DNA was detected during the first 2 h of germination. The addition of ethidium bromide (final concentration, 0.35 μg/ml) inhibited DNA repair during early outgrowth. Increased sensitivity of spores to heat after pretreatment with sublethal concentrations of ethidium bromide was due to the inhibition of the repair of heat-damaged DNA.
PMCID: PMC241736  PMID: 16346805
3.  Morphogenesis of Bacillus Spore Surfaces 
Journal of Bacteriology  2003;185(21):6255-6261.
Spores produced by bacilli are encased in a proteinaceous multilayered coat and, in some species (including Bacillus anthracis), further surrounded by a glycoprotein-containing exosporium. To characterize bacillus spore surface morphology and to identify proteins that direct formation of coat surface features, we used atomic-force microscopy (AFM) to image the surfaces of wild-type and mutant spores of Bacillus subtilis, as well as the spore surfaces of Bacillus cereus 569 and the Sterne strain of Bacillus anthracis. This analysis revealed that the coat surfaces in these strains are populated by a series of bumps ranging between 7 and 40 nm in diameter, depending on the species. Furthermore, a series of ridges encircled the spore, most of which were oriented along the long axis of the spore. The structures of these ridges differ sufficiently between species to permit species-specific identification. We propose that ridges are formed early in spore formation, when the spore volume likely decreases, and that when the spore swells during germination the ridges unfold. AFM analysis of a set of B. subtilis coat protein gene mutants revealed three coat proteins with roles in coat surface morphology: CotA, CotB, and CotE. Our data indicate novel roles for CotA and CotB in ridge pattern formation. Taken together, these results are consistent with the view that the coat is not inert. Rather, the coat is a dynamic structure that accommodates changes in spore volume.
PMCID: PMC219407  PMID: 14563859
4.  Combined Effects of Ultrahigh Vacuum and Temperature on the Viability of Some Spores and Soil Organisms1 
Applied Microbiology  1963;11(3):202-210.
Considerably fewer spores of Bacillus stearothermophilus, B. megaterium, and Clostridium sporogenes were recovered than were spores of B. subtilis var. niger and Aspergillus niger after 4 to 5 days at 53 and 60 C in ultrahigh vacuum. There were no significant differences in the recoveries of these five organisms at 25 C and atmospheric pressure, and after exposure to 25 and -190 C in vacuum. At 60 C, a far greater decrease in viability was demonstrated for B. stearothermophilus, B. megaterium, and C. sporogenes in ultrahigh vacuum than at atmospheric pressure. Viable B. subtilis var. niger spores were not detected in an initial 107 spores after retention at 90 C and ultrahigh vacuum, and 104 spores were viable after 5 days at 90 C and atmospheric pressure from an initial 106 spores. Molds and actinomycetes in soil were particularly resistant up to 69 C in vacuum. Actinomycetes were the only soil organisms recovered so far at 120 C.
PMCID: PMC1057974  PMID: 14025505
5.  Multifactorial Resistance of Bacillus subtilis Spores to High-Energy Proton Radiation: Role of Spore Structural Components and the Homologous Recombination and Non-Homologous End Joining DNA Repair Pathways 
Astrobiology  2012;12(11):1069-1077.
The space environment contains high-energy charged particles (e.g., protons, neutrons, electrons, α-particles, heavy ions) emitted by the Sun and galactic sources or trapped in the radiation belts. Protons constitute the majority (87%) of high-energy charged particles. Spores of Bacillus species are one of the model systems used for astro- and radiobiological studies. In this study, spores of different Bacillus subtilis strains were used to study the effects of high energetic proton irradiation on spore survival. Spores of the wild-type B. subtilis strain [mutants deficient in the homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair pathways and mutants deficient in various spore structural components such as dipicolinic acid (DPA), α/β-type small, acid-soluble spore protein (SASP) formation, spore coats, pigmentation, or spore core water content] were irradiated as air-dried multilayers on spacecraft-qualified aluminum coupons with 218 MeV protons [with a linear energy transfer (LET) of 0.4 keV/μm] to various final doses up to 2500 Gy. Spores deficient in NHEJ- and HR-mediated DNA repair were significantly more sensitive to proton radiation than wild-type spores, indicating that both HR and NHEJ DNA repair pathways are needed for spore survival. Spores lacking DPA, α/β-type SASP, or with increased core water content were also significantly more sensitive to proton radiation, whereas the resistance of spores lacking pigmentation or spore coats was essentially identical to that of the wild-type spores. Our results indicate that α/β-type SASP, core water content, and DPA play an important role in spore resistance to high-energy proton irradiation, suggesting their essential function as radioprotectants of the spore interior. Key Words: Bacillus—Spores—DNA repair—Protection—High-energy proton radiation. Astrobiology 12, 1069–1077.
PMCID: PMC3491616  PMID: 23088412
6.  Ultrastructural Analysis During Germination and Outgrowth of Bacillus subtilis Spores 
Journal of Bacteriology  1974;120(1):475-481.
Electron microscopy of thin sections of dormant and germinating spores of Bacillus subtilis 168 revealed a progressive change in the structure of the cortex, outer spore coat, and inner spore coat. The initial changes were observed in the cortex region, which showed a loose fibrous network within 10 min of germination, and in the outer spore coat, which began to be sloughed off. The permeability of the complex outer spore layers was modified within 10 min, since, at this time, the internal structures of the spore coat were readily stainable. A nicking degradation action of the laminated inner spore coat began at 20 min, and this progressed for the next 20 min leading to the loosening of the inner spore coat. By 30 min, the outer spore coat showed signs of disintegration, and at 40 min, both the outer and inner spore coats were degraded extensively. At 30 to 40 min, a period just preceding net deoxyribonucleic acid synthesis, mesosomes became very prominent in the inner spore core and the cell wall began to thicken around the spore core. At 50 min, an emerging cell was observed, and by 60 min, there was clear evidence for elongation of the emerging cell and the presence of two nuclear bodies. At 90 min, elongation had been followed by the first cell division. There was evidence for spore coat fragments at the opposite poles of the dividing cell.
PMCID: PMC245785  PMID: 4213660
7.  Germination properties of a spore coat-defective mutant of Bacillus subtilis. 
Journal of Bacteriology  1981;146(3):1106-1116.
The presence of the gerE36 mutation in strains of Bacillus subtilis 168 resulted in poor germination of their spores in a range of germinants, as measured by the fall in absorbance of spore suspensions. Although resistant to heat and organic solvents, spores were sensitive to lysozyme; electron microscopy revealed that their coat structure was incomplete. These spores responded to germinants by losing heat resistance and changing from phase bright to phase gray. The release of dipicolinic acid and the fall in absorbance of spore suspensions reached only 75 and 50% of wild-type levels, respectively, but followed the same time course as the loss of heat resistance. Although the germination response was incomplete, the concentration of L-alanine required to elicit it was the same for the mutant as for the wild type. The properties of mutant spores suggest that an intact spore coat is not required for the initial interaction between germinant and spore, but that the coat layers may contain molecules important in later stages of germination. In transduction with phage SPP1, the gerE36 mutation mapped between citF and ilvB and was 90% cotransduced with citF2. The gerE mutation identifies the location of a gene important for the progress of late stages of spore formation.
PMCID: PMC216967  PMID: 6787012
8.  Roles of DacB and Spm Proteins in Clostridium perfringens Spore Resistance to Moist Heat, Chemicals, and UV Radiation▿  
Applied and Environmental Microbiology  2008;74(12):3730-3738.
Clostridium perfringens food poisoning is caused mainly by enterotoxigenic type A isolates that typically possess high spore heat resistance. Previous studies have shown that α/β-type small, acid-soluble proteins (SASP) play a major role in the resistance of Bacillus subtilis and C. perfringens spores to moist heat, UV radiation, and some chemicals. Additional major factors in B. subtilis spore resistance are the spore's core water content and cortex peptidoglycan (PG) structure, with the latter properties modulated by the spm and dacB gene products and the sporulation temperature. In the current work, we have shown that the spm and dacB genes are expressed only during C. perfringens sporulation and have examined the effects of spm and dacB mutations and sporulation temperature on spore core water content and spore resistance to moist heat, UV radiation, and a number of chemicals. The results of these analyses indicate that for C. perfringens SM101 (i) core water content and, probably, cortex PG structure have little if any role in spore resistance to UV and formaldehyde, presumably because these spores’ DNA is saturated with α/β-type SASP; (ii) spore resistance to moist heat and nitrous acid is determined to a large extent by core water content and, probably, cortex structure; (iii) core water content and cortex PG cross-linking play little or no role in spore resistance to hydrogen peroxide; (iv) spore core water content decreases with higher sporulation temperatures, resulting in spores that are more resistant to moist heat; and (v) factors in addition to SpmAB, DacB, and sporulation temperature play roles in determining spore core water content and thus, spore resistance to moist heat.
PMCID: PMC2446547  PMID: 18441110
9.  Cloning of a small, acid-soluble spore protein gene from Bacillus subtilis and determination of its complete nucleotide sequence. 
Journal of Bacteriology  1985;161(1):333-339.
The first Bacillus subtilis small, acid-soluble spore protein (SASP) gene has been cloned by using previously cloned B. megaterium SASP genes as DNA-DNA hybridization probes. Determination of the DNA sequence of the B. subtilis SASP gene showed that it codes for a 72-residue protein (termed SASP-1) containing a single spore protease cleavage site as well as other sequences conserved in Bacillus megaterium SASPs A, C, C-1, C-2, and C-3. The B. subtilis SASP-1 genes's coding sequence is preceded by a potential Bacillus ribosome-binding site, and is followed by a sequence that could form a stem-and-loop structure characteristic of transcription termination sites. Upstream from the coding sequence there are no obvious homologies with other B. subtilis sporulation genes, but similarities with B. megaterium SASP genes are evident. SASP-1 mRNA (290 bases long) is absent from vegetative cells, but appears midway in sporulation and then disappears. The cloned SASP-1 gene hybridizes to three bands other than the SASP-1 gene itself in EcoRI or HindIII digests of B. subtilis DNA. Presumably these other bands represent SASP genes related to the SASP-1 gene, and we have been able to detect at least three such proteins in B. subtilis spores.
PMCID: PMC214876  PMID: 2981806
10.  Proteins Involved in Formation of the Outermost Layer of Bacillus subtilis Spores ▿† 
Journal of Bacteriology  2011;193(16):4075-4080.
To investigate the outermost structure of the Bacillus subtilis spore, we analyzed the accessibility of antibodies to proteins on spores of B. subtilis. Anti-green fluorescent protein (GFP) antibodies efficiently accessed GFP fused to CgeA or CotZ, which were previously assigned to the outermost layer termed the spore crust. However, anti-GFP antibodies did not bind to spores of strains expressing GFP fused to 14 outer coat, inner coat, or cortex proteins. Anti-CgeA antibodies bound to spores of wild-type and CgeA-GFP strains but not cgeA mutant spores. These results suggest that the spore crust covers the spore coat and is the externally exposed, outermost layer of the B. subtilis spore. We found that CotZ was essential for the spore crust to surround the spore but not for spore coat formation, indicating that CotZ plays a critical role in spore crust formation. In addition, we found that CotY-GFP was exposed on the surface of the spore, suggesting that CotY is an additional component of the spore crust. Moreover, the localization of CotY-GFP around the spore depended on CotZ, and CotY and CotZ depended on each other for spore assembly. Furthermore, a disruption of cotW affected the assembly of CotV-GFP, and a disruption of cotX affected the assembly of both CotV-GFP and CgeA-GFP. These results suggest that cgeA and genes in the cotVWXYZ cluster are involved in spore crust formation.
PMCID: PMC3147665  PMID: 21665972
11.  Antimicrobial Effects of Interferon-Inducible CXC Chemokines against Bacillus anthracis Spores and Bacilli▿  
Infection and Immunity  2009;77(4):1664-1678.
Based on previous studies showing that host chemokines exert antimicrobial activities against bacteria, we sought to determine whether the interferon-inducible Glu-Leu-Arg-negative CXC chemokines CXCL9, CXCL10, and CXCL11 exhibit antimicrobial activities against Bacillus anthracis. In vitro analysis demonstrated that all three CXC chemokines exerted direct antimicrobial effects against B. anthracis spores and bacilli including marked reductions in spore and bacillus viability as determined using a fluorometric assay of bacterial viability and CFU determinations. Electron microscopy studies revealed that CXCL10-treated spores failed to undergo germination as judged by an absence of cytological changes in spore structure that occur during the process of germination. Immunogold labeling of CXCL10-treated spores demonstrated that the chemokine was located internal to the exosporium in association primarily with the spore coat and its interface with the cortex. To begin examining the potential biological relevance of chemokine-mediated antimicrobial activity, we used a murine model of inhalational anthrax. Upon spore challenge, the lungs of C57BL/6 mice (resistant to inhalational B. anthracis infection) had significantly higher levels of CXCL9, CXCL10, and CXCL11 than did the lungs of A/J mice (highly susceptible to infection). Increased CXC chemokine levels were associated with significantly reduced levels of spore germination within the lungs as determined by in vivo imaging. Taken together, our data demonstrate a novel antimicrobial role for host chemokines against B. anthracis that provides unique insight into host defense against inhalational anthrax; these data also support the notion for an innovative approach in treating B. anthracis infection as well as infections caused by other spore-forming organisms.
PMCID: PMC2663164  PMID: 19179419
12.  Bacillus subtilis spore coats: complexity and purification of a unique polypeptide component. 
Journal of Bacteriology  1978;135(3):1091-1106.
Extensively washed, dormant spores of Bacillus subtilis were disrupted with glass beads in buffer at pH 7 in the presence of protease inhibitors. Approximately 31% of the total spore protein was soluble, and another 14% was removed from the insoluble fraction by hydrolysis with lysozyme and washing with 1 M KCl and 0.1% sodium dodecyl sulfate. The residual spore integuments comprised 55% of the total spore proteins and consisted of coats and residual membrane components. Treatment of integuments with sodium dodecyl sulfate and reducing agents at pH 10 solubilized 40% of the total spore protein. Seven low-molecular-weight polypeptide components of this solubilized fraction comprised 27% of the total spore protein. They are not normal membrane components and reassociated to form fibrillar structures resembling spore coat fragments. The residual insoluble material (15% of the total spore protein) was rich in cysteine and was probably also derived from the spore coats. A solubilized coat polypeptide of molecular weight 12,200 has been purified in good yield (4 to 5% of the total spore protein). Five amino acids account for 92% of its total amino acid residues: glycine, 19%; tyrosine, 31%; proline, 23%; arginine, 13%; and phenylalanine, 6%.
PMCID: PMC222486  PMID: 99427
13.  Prevention of DNA damage in spores and in vitro by small, acid-soluble proteins from Bacillus species. 
Journal of Bacteriology  1993;175(5):1367-1374.
The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP). These proteins alter DNA structure in vivo and in vitro, providing spore resistance to UV light. In addition, heat treatments (e.g., 85 degrees C for 30 min) which give little killing of wild-type spores of B. subtilis kill > 99% of spores which lack most alpha/beta-type SASP (termed alpha - beta - spores). Similar large differences in survival of wild-type and alpha - beta - spores were found at 90, 80, 65, 22, and 10 degrees C. After heat treatment (85 degrees C for 30 min) or prolonged storage (22 degrees C for 6 months) that gave > 99% killing of alpha - beta - spores, 10 to 20% of the survivors contained auxotrophic or asporogenous mutations. However, alpha - beta - spores heated for 30 min at 85 degrees C released no more dipicolinic acid than similarly heated wild-type spores (< 20% of the total dipicolinic acid) and triggered germination normally. In contrast, after a heat treatment (93 degrees C for 30 min) that gave > or = 99% killing of wild-type spores, < 1% of the survivors had acquired new obvious mutations, > 85% of the spore's dipicolinic acid had been released, and < 1% of the surviving spores could initiate spore germination. Analysis of DNA extracted from heated (85 degrees C, 30 min) and unheated wild-type spores and unheated alpha - beta - spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA. In contrast, the DNA from heated alpha- beta- spores had more than 10 single-strand breaks per 20 kb. These data suggest that binding of alpha/beta-type SASP to spore DNA in vivo greatly reduces DNA damage caused by heating, increasing spore heat resistance and long-term survival. While the precise nature of the initial DNA damage after heating of alpha- beta- spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination. A role for alpha/beta-type SASP in protecting DNA against depurination (and thus promoting spore survival) was further suggested by the demonstration that these proteins reduce the rate of DNA depurination in vitro at least 20-fold.
PMCID: PMC193223  PMID: 8444799
14.  Dynamics of Spore Coat Morphogenesis in Bacillus subtilis 
Molecular Microbiology  2011;83(2):245-260.
Spores of Bacillus subtilis are encased in a protective coat made up of at least 70 proteins. The structure of the spore coat has been examined using a variety of genetic, imaging and biochemical techniques, however, the majority of these studies have focused on mature spores. In this study we use a library of 41 spore coat proteins fused to the Green Fluorescent Protein (GFP) to examine spore coat morphogenesis over the time-course of sporulation. We found considerable diversity in the localization dynamics of coat proteins and were able to establish 6 classes based on localization kinetics. Localization dynamics correlate well with the known transcriptional regulators of coat gene expression. Previously, we described the existence of multiple layers in the mature spore coat. Here, we find that the spore coat initially assembles a scaffold that is organized into multiple layers on one pole of the spore. The coat then encases the spore in multiple coordinated waves. Encasement is driven, at least partially, by transcription of coat genes and deletion of sporulation transcription factors arrests encasement. We also identify the trans-compartment SpoIIIAH-SpoIIQ channel as necessary for encasement. This is the first demonstration of a forespore contribution to spore coat morphogenesis.
PMCID: PMC3256263  PMID: 22171814
Spore coat; Bacillus subtilis; sporulation; morphogenesis; spore crust
15.  Effect of Microwaves on Escherichia coli and Bacillus subtilis 
Applied Microbiology  1967;15(6):1371-1375.
Suspensions of Escherichia coli and Bacillus subtilis spores were exposed to conventional thermal and microwave energy at 2,450 MHz. The degrees of inactivation by the different energy sources were compared quantitatively. During the transient heating period by microwave energy, approximately a 6 log cycle reduction in viability was encountered for E. coli. This reduction was nearly identical to what is expected for the same time-temperature exposure to conventional heating. Heating of B. subtilis spores by conventional and microwave energy was also carried out at 100 C, in ice and for transient heating. The degree of inactivation by microwave energy was again identical to that by conventional heating. In conclusion, inactivation of E. coli and B. subtilis by exposure to microwaves is solely due to the thermal energy, and there is no per se effect of microwaves.
PMCID: PMC547203  PMID: 16349747
16.  UV Resistance of Bacillus anthracis Spores Revisited: Validation of Bacillus subtilis Spores as UV Surrogates for Spores of B. anthracis Sterne 
Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.
PMCID: PMC143644  PMID: 12571068
17.  Morphogenesis of the Bacillus anthracis Spore▿  
Journal of Bacteriology  2006;189(3):691-705.
Bacillus spp. and Clostridium spp. form a specialized cell type, called a spore, during a multistep differentiation process that is initiated in response to starvation. Spores are protected by a morphologically complex protein coat. The Bacillus anthracis coat is of particular interest because the spore is the infective particle of anthrax. We determined the roles of several B. anthracis orthologues of Bacillus subtilis coat protein genes in spore assembly and virulence. One of these, cotE, has a striking function in B. anthracis: it guides the assembly of the exosporium, an outer structure encasing B. anthracis but not B. subtilis spores. However, CotE has only a modest role in coat protein assembly, in contrast to the B. subtilis orthologue. cotE mutant spores are fully virulent in animal models, indicating that the exosporium is dispensable for infection, at least in the context of a cotE mutation. This has implications for both the pathophysiology of the disease and next-generation therapeutics. CotH, which directs the assembly of an important subset of coat proteins in B. subtilis, also directs coat protein deposition in B. anthracis. Additionally, however, in B. anthracis, CotH effects germination; in its absence, more spores germinate than in the wild type. We also found that SpoIVA has a critical role in directing the assembly of the coat and exosporium to an area around the forespore. This function is very similar to that of the B. subtilis orthologue, which directs the assembly of the coat to the forespore. These results show that while B. anthracis and B. subtilis rely on a core of conserved morphogenetic proteins to guide coat formation, these proteins may also be important for species-specific differences in coat morphology. We further hypothesize that variations in conserved morphogenetic coat proteins may play roles in taxonomic variation among species.
PMCID: PMC1797280  PMID: 17114257
18.  Structure-Based Functional Studies of the Effects of Amino Acid Substitutions in GerBC, the C Subunit of the Bacillus subtilis GerB Spore Germinant Receptor ▿† 
Journal of Bacteriology  2011;193(16):4143-4152.
Highly conserved amino acid residues in the C subunits of the germinant receptors (GRs) of spores of Bacillus and Clostridium species have been identified by amino acid sequence comparisons, as well as structural predictions based on the high-resolution structure recently determined for the C subunit of the Bacillus subtilis GerB GR (GerBC). Single and multiple alanine substitutions were made in these conserved residues in three regions of GerBC, and the effects of these changes on B. subtilis spore germination via the GerB GR alone or in concert with the GerK GR, as well as on germination via the GerA GR, were determined. In addition, levels of the GerBC variants in the spore inner membrane were measured, and a number of the GerBC proteins were expressed and purified and their solubility and aggregation status were assessed. This work has done the following: (i) identified a number of conserved amino acids that are crucial for GerBC function in spore germination via the GerB GR and that do not alter spores' levels of these GerBC variants; (ii) identified other conserved GerBC amino acid essential for the proper folding of the protein and/or for assembly of GerBC in the spore inner membrane; (iii) shown that some alanine substitutions in GerBC significantly decrease the GerA GR's responsiveness to its germinant l-valine, consistent with there being some type of interaction between GerA and GerB GR subunits in spores; and (iv) found no alanine substitutions that specifically affect interaction between the GerB and GerK GRs.
PMCID: PMC3147670  PMID: 21685283
19.  Crystallization and preliminary X-ray analysis of the complex between a Bacillus subtilis α/β-type small acid-soluble spore protein and DNA 
An α/β-type small, acid-soluble spore protein (SASP) from Bacillus subtilis, a major source of DNA protection against damaging effects in spores, was crystallized in a functionally relevant complex with a double-stranded DNA. This report provides insights into initial characterization of the complex and its structure elucidation.
An engineered variant of an α/β-type small acid-soluble spore protein (SASP) from Bacillus subtilis was crystallized in a complex with a ten-base-pair double-stranded DNA by the hanging-drop vapor-diffusion method using ammonium sulfate as a precipitating agent. Crystals grew at 281 K using sodium cacodylate buffer pH 5.5 and these crystals diffracted X-rays to beyond 2.4 Å resolution using synchrotron radiation. The crystallized complex contains two or three SASP molecules bound to one DNA molecule. The crystals belong to the hexagonal space group P6122 or P6522, with unit-cell parameters a = b = 87.0, c = 145.4 Å, α = β = 90.0, γ = 120.0°. Diffraction data were 96.6% complete to 2.4 Å resolution, with an R sym of 8.5%. Structure solution by the multiwavelength/single-wavelength anomalous dispersion method using isomorphous crystals of selenomethionine-labeled protein is in progress.
PMCID: PMC2335083  PMID: 17554173
small acid-soluble spore protein; spore resistance; DNA; Bacillus subtilis
20.  Bacillus subtilis Diacylglycerol Kinase (DgkA) Enhances Efficient Sporulation 
Journal of Bacteriology  2003;185(17):5306-5309.
The sn-1,2-diacylglycerol kinase homologue gene, dgkA, is a sporulation gene indispensable for the maintenance of spore stability and viability in Bacillus subtilis. After 6 h of growth in resuspension medium, the endospore morphology of the dgkA mutant by standard phase-contrast microscopy was normal; however, after 9 h, the endospores appeared mostly dark by phase-contrast microscopy, suggesting a defect in the spores. Moreover, electron microscopic studies revealed an abnormal cortex structure in mutant endospores 6 h after the onset of sporulation, an indication of cortex degeneration. In addition, a significant decrease in the dipicolinic acid content of mutant spores was observed. We also found that dgkA is expressed mainly during the vegetative phase. It seems likely that either the DgkA produced during growth prepares the cell for an essential step in sporulation or the enzyme persists into sporulation and performs an essential function.
PMCID: PMC180973  PMID: 12923107
21.  Monitoring the Wet-Heat Inactivation Dynamics of Single Spores of Bacillus Species by Using Raman Tweezers, Differential Interference Contrast Microscopy, and Nucleic Acid Dye Fluorescence Microscopy▿  
Applied and Environmental Microbiology  2011;77(14):4754-4769.
Dynamic processes during wet-heat treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis at 80 to 90°C were investigated using dual-trap Raman spectroscopy, differential interference contrast (DIC) microscopy, and nucleic acid stain (SYTO 16) fluorescence microscopy. During spore wet-heat treatment, while the spores' 1:1 chelate of Ca2+ with dipicolinic acid (CaDPA) was released rapidly at a highly variable time Tlag, the levels of spore nucleic acids remained nearly unchanged, and the Tlag times for individual spores from the same preparation were increased somewhat as spore levels of CaDPA increased. The brightness of the spores' DIC image decreased by ∼50% in parallel with CaDPA release, and there was no spore cortex hydrolysis observed. The lateral diameters of the spores' DIC image and SYTO 16 fluorescence image also decreased in parallel with CaDPA release. The SYTO 16 fluorescence intensity began to increase during wet-heat treatment at a time before Tlag and reached maximum at a time slightly later than Trelease. However, the fluorescence intensities of wet-heat-inactivated spores were ∼15-fold lower than those of nutrient-germinated spores, and this low SYTO 16 fluorescence intensity may be due in part to the low permeability of the dormant spores' inner membranes to SYTO 16 and in part to nucleic acid denaturation during the wet-heat treatment.
PMCID: PMC3147409  PMID: 21602365
22.  Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination. 
Journal of Bacteriology  1992;174(3):807-814.
During germination of spores of Bacillus species the degradation of the spore's pool of small, acid-soluble proteins (SASP) is initiated by a protease termed GPR, the product of the gpr gene. Bacillus megaterium and B. subtilis mutants with an inactivated gpr gene grew, sporulated, and triggered spore germination as did gpr+ strains. However, SASP degradation was very slow during germination of gpr mutant spores, and in rich media the time taken for spores to return to vegetative growth (defined as outgrowth) was much longer in gpr than in gpr+ spores. Not surprisingly, gpr spores had much lower rates of RNA and protein synthesis during outgrowth than did gpr+ spores, although both types of spores had similar levels of ATP. The rapid decrease in the number of negative supertwists in plasmid DNA seen during germination of gpr+ spores was also much slower in gpr spores. Additionally, UV irradiation of gpr B. subtilis spores early in germination generated significant amounts of spore photoproduct and only small amounts of thymine dimers (TT); in contrast UV irradiation of germinated gpr+ spores generated almost no spore photoproduct and three to four times more TT. Consequently, germinated gpr spores were more UV resistant than germinated gpr+ spores. Strikingly, the slow outgrowth phenotype of B. subtilis gpr spores was suppressed by the absence of major alpha/beta-type SASP. These data suggest that (i) alpha/beta-type SASP remain bound to much, although not all, of the chromosome in germinated gpr spores; (ii) the alpha/beta-type SASP bound to the chromosome in gpr spores alter this DNA's topology and UV photochemistry; and (iii) the presence of alpha/beta-type SASP on the chromosome is detrimental to normal spore outgrowth.
PMCID: PMC206157  PMID: 1732215
23.  Role of Dipicolinic Acid in the Germination, Stability, and Viability of Spores of Bacillus subtilis▿  
Journal of Bacteriology  2008;190(14):4798-4807.
Spores of Bacillus subtilis spoVF strains that cannot synthesize dipicolinic acid (DPA) but take it up during sporulation were prepared in medium with various DPA concentrations, and the germination and viability of these spores as well as the DPA content in individual spores were measured. Levels of some other small molecules in DPA-less spores were also measured. These studies have allowed the following conclusions. (i) Spores with no DPA or low DPA levels that lack either the cortex-lytic enzyme (CLE) SleB or the receptors that respond to nutrient germinants could be isolated but were unstable and spontaneously initiated early steps in spore germination. (ii) Spores that lacked SleB and nutrient germinant receptors and also had low DPA levels were more stable. (iii) Spontaneous germination of spores with no DPA or low DPA levels was at least in part via activation of SleB. (iv) The other redundant CLE, CwlJ, was activated only by the release of high levels of DPA from spores. (v) Low levels of DPA were sufficient for the viability of spores that lacked most α/β-type small, acid-soluble spore proteins. (vi) DPA levels accumulated in spores prepared in low-DPA-containing media varied greatly between individual spores, in contrast to the presence of more homogeneous DPA levels in individual spores made in media with high DPA concentrations. (vii) At least the great majority of spores of several spoVF strains that contained no DPA also lacked other major spore small molecules and had gone through some of the early reactions in spore germination.
PMCID: PMC2446992  PMID: 18469099
24.  Influence of environmental storage relative humidity on biological indicator resistance, viability, and moisture content. 
The effect of environmental storage relative humidity (RH) on the moisture content, viability, and moist heat and gaseous ethylene oxide (EO) resistance of biological indicators (BIs) was evaluated. No statistically significant difference was observed between the initial Bacillus stearothermophilus spore population and the spore population of BIs stored at 20 degrees C and 0, 20, 44, of 55% RH or under ambient, 4 degrees C, or -20 degrees C conditions after 12 months. A statistically significant decrease in moist heat resistance from initial starting levels was found for BIs stored at 20 degrees C and either 0 or 20% RH. There was a statistically significant decrease in the B. subtilis BI spore population, compared with initial levels, when the BIs were stored at 20 degrees C and 0% RH concomitant with a significant increase in their EO resistance. BI storage at 20 degrees C and 20 or 44% RH, or under ambient, 4 degrees C, or -20 degrees C conditions, had no significant effect on EO resistance. BIs stored at 20 degrees C and 66% RH demonstrated a significantly lower EO resistance compared with starting levels.
PMCID: PMC241882  PMID: 7073275
25.  Bacillus subtilis Spores as Vaccine Adjuvants: Further Insights into the Mechanisms of Action 
PLoS ONE  2014;9(1):e87454.
Bacillus subtilis spores have received growing attention regarding potential biotechnological applications, including the use as probiotics and in vaccine formulations. B. subtilis spores have also been shown to behave as particulate vaccine adjuvants, promoting the increase of antibody responses after co-administration with antigens either admixed or adsorbed on the spore surface. In this study, we further evaluated the immune modulatory properties of B. subtilis spores using a recombinant HIV gag p24 protein as a model antigen. The adjuvant effects of B. subtilis spores were not affected by the genetic background of the mouse lineage and did not induce significant inflammatory or deleterious effects after parenteral administration. Our results demonstrated that co-administration, but not adsorption to the spore surface, enhanced the immunogenicity of that target antigen after subcutaneous administration to BALB/c and C57BL/6 mice. Spores promoted activation of antigen presenting cells as demonstrated by the upregulation of MHC and CD40 molecules and enhanced secretion of pro-inflammatory cytokines by murine dendritic cells. In addition, in vivo studies indicated a direct role of the innate immunity on the immunomodulatory properties of B. subtilis spores, as demonstrated by the lack of adjuvant effects on MyD88 and TLR2 knockout mouse strains.
PMCID: PMC3903701  PMID: 24475289

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